Installation

This can be installed from the commandline using cargo.

# installing from crates.io
cargo install sgcount

# installing from github
git clone https://github.com/noamteyssier/sgcount
cd sgcount
cargo install --path .

Usage

This is meant to be used as a commandline tool and it expects at minimum two files.

The first is the Library, which is a fasta formatted file describing the sequencing library that the sequences should be aligned to.

The second is the Sequencing, which is generally a fastq or fastq.gz file representing the sequencing results of a CRISPRi/a screen.

Running sgcount

Basic Usage

The experiment can then be run using the commandline interface.

sgcount -l <path_to_library> -i <path_to_sequencing>

Multiple Sequencing Files

If you have multiple files to count you can provide them as extra arguments to the -i flag.

sgcount -l <path_to_library> -i <path_to_sequencing_a> <path_to_sequencing_b>

Providing Sample Names

If you have a shorthand alias for your sample names you can provide them with the -n flag. Note that the number of sample names must be equal to the number of provided samples (the program will quit otherwise).

sgcount -l <path_to_library> -i <path_to_sequencing_a> <path_to_sequencing_b> -n <name_a> <name_b>

Providing a Gene -> sgRNA Mapping

If you would like to associate each sgRNA with its parent gene you can provide that mapping with the -g flag. This will expect a two column tab-delim file. The first column will be the gene, and the second will be the sgRNA name (not sequence). This will be validated against the library - and any sgRNAs that cannot be mapped against this table in the library will cause the program to quit.

If your library is fasta formatted and the gene is provided in the header you can generate this file easily using my tool sgrna-table within fxtools.

sgcount -l <path_to_library> -i <path_to_sequencing> -g g2s.txt

Setting the Offset

The program will automatically determine the offset if this flag is not provided. However, if you know the offset a priori you can supply it with the -a flag. If you recover all zeros in your counts it is recommended to not supply this flag and let the program determine the offset algorithmically.

For example, if you have a 10bp adapter sequence before the sgRNA then you can run the following:

sgcount -l <path_to_library> -i <path_to_sequencing> -a 10

Disallowing Mismatches

By default sgcount will allow single mismatches, but if you would like to have only exact matches you can specify it with the -x flag.

sgcount -l <path_to_library> -i <path_to_sequencing> -x

Setting the Output File

By default sgcount will write the results to stdout, but if you'd like to specify the output path directly you can do so with the -o flag.

sgcount -l <path_to_library> -i <path_to_sequencing> -o <path_to_output>

Shutting off verbosity

By default the program will show how much time is being elapsed as well as update the user on its progress. You can shut off this behavior with the -q flag.

sgcount -l <path_to_library> -i <path_to_sequencing> -q

Example Fasta Library

An example library is provided in example/library.fa

>lib.0
ATAGCCCGGCGGTCTGCTGG
>lib.1
TAAGGCACTATAGCAATGAG
>lib.2
GTAGATAAAACGTGTGGCCC
>lib.3
TTCATACAATAACGACGTGC
>lib.4
AAGGCGACCATCTACCCTTG
>lib.5
CGCATAAACCCTTTCAACTG
>lib.6
GGAGTGGAGCGCTGAGTCGT
>lib.7
GGTAAGTACACATCGCCATG
>lib.8
CAGGTAGGACTACAGAGCTG
>lib.9
GCCTATGGTTGGTAGGCAAG
>lib.10
CGGGGCGTGCTATACGCATT

Example Sequencing

An example of some sequencing data is provided in example/sequence.fq

@seq.AACGTTCTCCAGTATGAAAG.0
ATNGCAACGTTCTCCAGTATGAAAGTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
43212322322242515413324331541432414553224213511111344532442224113253532413451225
@seq.CGGTTCCCTGCCGCTACGAG.1
ATNGCCGGTTCCCTGCCGCTACGAGTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
23233555242215242532355415123114534342422111212445152424453152255425331534444213
@seq.CTCGCCGCGCGGCACTATTG.2
ATNGCCTCGCCGCGCGGCACTATTGTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
54532443112431133412311213532322244241224451345215242125451241523232121145343513
@seq.TATAGACATATTATACGTCC.3
ATNGCTATAGACATATTATACGTCCTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
33231435244335232142144245314521453354531535215154523311555133141253412544112225
@seq.GGTTTGTTACGCGAGCAGTT.4
ATNGCGGTTTGTTACGCGAGCAGTTTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
52245315235112214142511531543122452153335313154325215245554114252235434421423233
@seq.ATACGCATCTTCGGAATGTA.5
ATNGCATACGCATCTTCGGAATGTATAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
31143423343255242141513351253142515145434443123244145415354115445255254212451244
@seq.AGGGTGCTTTTGATGTGGAT.6
ATNGCAGGGTGCTTTTGATGTGGATTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
53251513225425232352211233534114522215223524153354423322333521454544324423154421
@seq.CGCTCGCCTTCAAGCTACAT.7
ATNGCCGCTCGCCTTCAAGCTACATTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
44232545355144343215132235132555415544524212153151242432352221425555451214415433
@seq.ATCCGTTAACACCCGTGTAA.8
ATNGCATCCGTTAACACCCGTGTAATAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
11525225352243452555523453253222354324253121311522125114552224532131353125523242
@seq.TTTTCGAGATATCTTGCCTT.9
ATNGCTTTTCGAGATATCTTGCCTTTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
15352412434253544231322442452233153353522434245343321511215322155333313541233112
@seq.AACGTTCTCCAGTATGAAAG.10
ATNGCAACGTTCTCCAGTATGAAAGTAGCGACAAGACGGGCCAAGAGGGACTGCGCACCACGTAGTTACCCCGATCCTAT
+
24352514151524243135221555342112334153424555141234231424555513545151511254444444